The yield of recombinant proteins in plants determines their economic competitiveness as a production platform compared to microbes and mammalian cells. The promoter, untranslated regions (UTRs) and codon usage can all contribute to the yield, but potential interactions among these components have not been examined in detail. Here the effect of two promoters (35SS and nos) and four 5'UTRs on the spatiotemporal expression of DsRed mRNA and the accumulation of DsRed protein during transient expression in tobacco (Nicotiana tabacum) mediated by Agrobacterium tumefaciens is investigated. The authors found that the mRNA levels peaked 2-3 days post-infiltration (dpi), and rapidly declined thereafter, whereas DsRed protein was first detected after ≈3 days and concentrations continued to increase until at least 5 dpi. This temporal decoupling of mRNA and protein expression was strongest in the older leaves, which also produced the lowest DsRed yields. The accumulation of DsRed linearly correlated with mRNA levels in all but the youngest leaves, where more DsRed was synthesized per mRNA molecule. This was the case for both promoters, although the nos promoter had a higher protein/mRNA ratio than the 35SS promoter. Furthermore, the type of 5'UTR affected DsRed protein accumulation by 50% starting from similar levels of mRNA. The authors concluded that DsRed mRNA levels are not the limiting factor for DsRed protein expression in plants, but that translation-associated processes such as initiation, elongation, and release are bottlenecks that should be addressed in future studies.
Keywords: design of experiments; molecular farming; promoter/5′UTR combination; real time PCR; transient expression.
© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.